Immobilized Metal Ion Affinity Chromatography (IMAC) is one of the most frequently used techniques for purification of fusion proteins containing affinity sites for metal ions. IMAC is a separation principle that utilizes the differential affinity of proteins for immobilized metal ions to effect their separation. This differential affinity derives from the coordination bonds formed between metal ions and certain amino acid side chains exposed on the surface of the protein molecules. Since the interaction between the immobilized metal ions and the side chains of amino acids has a readily reversible character, it can be utilized for adsorption and then be disrupted using mild (i.e., non denaturing) conditions.
Adsorbents that are currently commercially available include iminodiacetic acid (IDA), nitriloacetic acid (NTA), caboxymethylated aspartic acid (CM-Asp), and tris-carboxymethyl ethylene diamine (TED). These ligands offer a maximum of tri- (IDA), tetra- (NTA, CM-Asp), and penta-dentate (TED) complexes with the respective metal ion. In most commercially available adsorbents, metal chelating ligands are provided at an average density of about 12 Å. Depending on the ligand, various metals can be chelated. Metal ions typically used in IMAC procedures have been classified into three categories—hard, intermediate, and soft—based on their preferential reactivity toward nucleophiles. The hard metal ions Fe3+, Ca2+, and Al3+ show a preference for oxygen; the soft metal ions Cu+, Hg2+, Ag+, and the like show a preference for sulfur; and intermediate metal ions such as Cu2+, Ni2+, Zn2+, and Co2+ coordinate nitrogen, oxygen, and sulfur. The number of cysteine residues on the surfaces of proteins is limited; therefore, histidine residues are the major targets for intermediate metal ions.
The observation that histidine residues bind to certain immobilized ions led to the development of histidine-containing “tags” for proteins to aid in purification of such proteins. In particular, peptide tags containing multiple histidines have been developed. For example hexa-histidine tags are commonly used with IMAC adsorbents for purification of recombinant proteins.
Despite the advances made in protein purification using IMAC, there is an ongoing need in the art for improved metal ion affinity tags for use in purifying proteins. The present invention addresses this need.
Literature
The following publications are of interest: Itakura, et al., Science 198:1056–63 (1977); Germino, et al., Proc. Natl. Acad. Sci. USA 80:6848–52 (1983); Nilsson et al., Nucleic Acids Res. 13:1151–62 (1985); Smith et al., Gene 32:321–27 (1984); Dobeli, et al., U.S. Pat. No. 5,284,933; Dobeli, et al., U.S. Pat. No. 5,310,663; U.S. Pat. No. 4,569,794; and U.S. Pat. No. 5,594,115.